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Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with β-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.

The transport of proteins and lipids between distinct cellular compartments is conducted by coated vesicles. These vesicles are formed by the self-assembly of coat proteins on a membrane, leading to collection of the vesicle cargo and membrane bending to form a bud. Scission at the bud neck releases the vesicle. X-ray crystallography and electron microscopy (EM) have recently generated models of isolated coat components and assembled coats. Here, we review these data to present a structural overview of the three main coats: clathrin, COPII, and COPI. The three coats have similar function, common ancestry, and structural similarities, but exhibit fundamental differences in structure and assembly. We describe the implications of structural similarities and differences for understanding the function, assembly principles, and evolution of vesicle coats., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Transmembrane proteins destined to endosomes are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles. The recognition of specific sequence motifs in transmembrane cargo by coated-pit proteins confers specificity on the endocytic process. Interaction of membrane cargo with the clathrin adaptor protein complex AP-2 is the major mechanism of cargo sorting into coated pits in mammalian cells. Recent studies have revealed a variety of alternative mechanisms of cargo recruitment involving additional adaptor proteins. These alternative mechanisms appear to be particularly important during clathrin-mediated endocytosis of signaling receptors.

Clathrin-mediated endocytosis is the major process by which transmembrane proteins are internalized from the cell's limiting membrane into the first compartment of the endosomal system, the early endosome. From here, these transmembrane cargo proteins, which are of widely varying type and function, are trafficked to their required destination. Endocytosis plays, therefore, an important role in cell signalling, nutrient uptake, cellular homoeostasis and the interaction of the cell with its external environment. The formation of clathrin-coated endocytic vesicles requires the complex interplay of many proteins with each other and with the membrane itself. Their formation has served as a paradigm for formation of all types of transport vesicle, which move cargo between the various membrane-bound compartments of the cell. Clathrin-coated vesicles (CCVs) possess three layers: the inner membrane layer, in which the transmembrane cargo is embedded, linked to the outer clathrin lattice by a layer of cargo-binding adaptors and proteins that aid and regulate vesicle formation. Protein X-ray crystallography in combination with biochemical, biophysical and cell biological assays has been used to investigate the structure and function of some of the proteins that make up the middle layer of CCVs. These proteins are diverse in their functions, but are all modular in nature, consisting of folded domains joined by long unstructured linkers. Within these linkers are short motifs that interact with the folded domains of other components of the CCV formation machinery. Many of these folded domains also bind directly to the membrane. These interactions, whose molecular basis we have studied, have affinities in the low micromolar range, making them readily reversible and easily regulated. The mechanism of CCV formation is discussed in the light of this structural and biochemical data.

The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

Clathrin coated vesicles were isolated from lactating rabbit mammary gland by differential centrifugation, centrifugation on (2)H2O-sucrose cushions and Sephacryl S-1000 chromatography. Mammary epithelial cells contain an unexpectedly high quantity of clathrin coated vesicles which appear heterogeneous in size, with a mean diameter of 95.9+/-10.5 nm and a density of 1.23 g x ml(-1). Analysis of clathrin coated vesicle adaptor composition by SDS-PAGE and western blot showed that only approximately 5-10% of total APs consist of AP-2 in isolated mammary gland clathrin coated vesicles whereas it represents approximately 70% of the total APs from bovine brain clathrin coated vesicles. Cargo molecules known to be transcytosed such as IgG, IgA, and the pIgR were detected in the clathrin coated vesicles, indicating that part of this vesicle population is involved in transcytotic pathways. However, as the vast majority of the clathrin coated vesicles contained AP-1, it was likely that these clathrin coated vesicles were involved in the secretory pathway. Relatively high quantities of furin and cation-independent mannose 6-phosphate receptor were detected in mammary clathrin coated vesicles. By immuno electron microscopy, AP-1 and the cation-independent mannose 6-phosphate receptor were localized in Golgi-associated vesicles and on the membrane of secretory vesicles. The presence of AP-1 in the coat patches on the membrane of secretory vesicles containing casein micelles, and the presence of alpha(s1)-casein in mammary gland clathrin coated vesicles, support a role for AP-1 in the maturation of secretory vesicles. Our data pinpoint the importance of clathrin coated vesicles in lactating mammary epithelial cells, and suggest these vesicles are involved in the transcytotic pathway, in sorting at the trans-Golgi network and in the biogenesis of casein-containing secretory vesicles.

The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.

Clathrin light chain B (LCB) is a major component of clathrin coated vesicles, which are structures involved in intracellular transport. A neuron-specific isoform of LCB is generated by incorporation of a single exon (EN) using an alternative splicing mechanism that reflects the special demands of neurons, such as axonal transport and synaptic neurotransmission. Here, we demonstrate that this neuron-specific exon is developmentally regulated and is excluded in non-neuronal cells because its 5' and 3' splice sites deviate from the mammalian consensus sequences. A gel retardation assay indicated the presence of a developmentally regulated factor in brain that binds to the neuronal exon. In addition, EN usage is repressed by increasing the concentration of htra2-beta1, a splice factor whose isoform expression is influenced by neuronal activity. We propose that a brain-specific factor is involved in EN recognition during development and adulthood. In addition, ubiquitously expressed splicing factors such as htra2-beta1 are involved in regulating EN expression in the adult brain., (Copyright 1999 Elsevier Science B.V.)

The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABAA receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [3H]muscimol. a GABAA-specific agonist. [3H]flunitrazepam a benzodiazepine agonist, and [35S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABAA receptor channels. Under standard conditions, the binding level of [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS to coated vesicles represented 12.3 +/- 1.8%, 7.9 +/- 1%, and 10.2 +/- 1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [3H]flunitrazepam binding site with a KD value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [3H]flunitrazepam and [35S]TBPS binding assays. For [3H]flunitrazepam binding to synaptic membranes, GABA gave an EC50 = 2.0 microM and at saturation (100 microM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [3H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [35S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABAA receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABAA receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110 +/- 14% and 29.5 +/- 3.8%, respectively, of the immunoreactivity for GABAA receptor beta 2 and alpha 1 subunits. Thus, in comparison to GABAA receptors on synaptic membranes, those on CCVs have a reduced alpha 1/beta 2-subunit ratio. It may be suggested that a selective decline in the content of alpha 1 subunits in coated vesicles could in part account for GABAA receptor uncoupling.

We have deduced the amino acid sequences of clathrin heavy chain (CHC) polypeptides based on cDNA and genomic clones from the mosquito, Aedes aegypti. Two isoforms which differ in the very beginning of the N-terminal domain, ovary-specific AaCHCa and somatic-specific AaCHCb, were identified, characterized and compared to one another as well as to CHC polypeptides from different species. The 1682 amino acid sequence of the AaCHCa isoform predicts a molecular mass (M[r]) of 191,743 daltons and an isoelectric point of 5.80, whereas the 1674 amino acid sequence of the AaCHCb isoform predicts a M(r) of 191,033 daltons and an isoelectric point of 5.71. Both mosquito AaCHC isoforms are highly conserved, with full-sequence identities of 88% to Drosophila melanogaster, 81% to mammal (rat, cow and human), 71% to C. elegans, 58% to Dictyostelium discoideum, and 49% to yeast CHC polypeptides. The highest degree of conservation is in the middle portion of the mosquito CHC molecule which includes the linker region and extended triskelion arm, with decreasing conservation through the N-terminal domain, trimerization domain, and the relatively diverged C-terminal region. The protein domains do not directly correspond to specific exons of the mosquito AaCHC gene, with the exception of exon 6 which encodes the C-terminal domain of the CHC polypeptide. Polyclonal antibodies raised against a bacteria-expressed AaCHC fusion protein recognized one major band of about 180 kDa in vitellogenic ovary whole-lysate. Immunogold labelling of the AaCHC polypeptide localized it to the coat of coated pits and coated vesicles in oocytes from vitellogenic follicles. Northern blot and in situ hybridization analyses suggest that regulation of AaCHC gene expression in the ovary is complex, and it likely involves both developmental and hormonal signals.

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.

We have previously isolated 3 different populations of clathrin coated vesicles (CCV) involved in the LDL-receptor traffic in bovine adrenal cortex. We now show that each CCV type contains the transferrin-R and the CI-MPR, therefore, they provide a good model for studying the membrane organization that may govern their targeting in one of the biosynthetic, endocytic and/or recycling pathways. Transferrin--prototype of recylcing ligand--, and alpha adaptin, dynamin and the 110 kDa phosphatidylinositol-3-kinase subunit--of the trafficking machinery--were mainly detected in only 2 of the vesicle populations which could be involved in the endocytic/recycling pathway. The third population which contained larger amounts of gamma adaptin and do not carry transferrin could be involved in the biosynthetic pathway. The vesicle lipid pattern and the saturation of their fatty acyl chains were analyzed and confirmed these results. The nature of the interactions between vesicle components was then determined using several classes of detergents. Only non ionic ones could solubilize the LDL-R in a complex with either alpha or gamma adaptin. In contrast, they dissociated clathrin or beta-beta' adaptins. Taken together these results prompt us to suggest an integrated model for targeting in membrane traffic. Besides specific targeting signals carried by cargo proteins and recognized by proteins from the coat and the cytosolic trafficking machinery, lipids would play a key modulatory role. At each step in the membrane traffic, the proteins which carry multiple targeting signals would interact transiently with a specific set of lipids. This would result in the exposure of the appropriate targeting signals which could now become recognized by the proper targeting machinery.

Clathrin-coated vesicles were isolated from soybean (Glycine max L.) cells in suspension culture and their purity was assessed using SDS-PAGE, peptide sequencing and electron microscopy. Antibodies raised to these coated vesicles were used to immunoscreen a soybean cDNA library in lambda gt11 and isolate a partial clone of the clathrin heavy chain (HC) gene. Full-length cDNA for soybean clathrin HC was deduced by 5' and 3' cDNA amplification. The cDNA encodes an amino acid sequence of 1,700 residues, which is slightly larger than rat clathrin HC and may account for the reduced mobility of plant clathrin on SDS-PAGE. Insertion of these extra residues is largely confined to the amino and carboxy termini. Other domains within the heavy chain arms, including those implicated in light chain binding and trimerisation, are relatively well conserved between eukaryotes. A computer algorithm to determine alpha-helical coiled-coil structures reveals that only one domain, aligning to residues 1,460-1,489 in rat clathrin HC, has a high probability for coiled-coil structure in all five eukaryotic clathrin HC sequences. This provides further evidence that the interaction between clathrin heavy and light chains is mediated by three bundles of coiled-coils near to the carboxy terminus. In analysing the role of plant clathrin in endocytotic trafficking, as against trafficking from the Golgi apparatus to the vacuole, our attention was focused on membrane recycling in tip-growing pollen tubes. These rapidly growing cells are highly secretory and require a high level of plasma membrane recycling to maintain the tube tip architecture. Monoclonal antibodies to plant clathrin HC confirmed that coated vesicles are relatively abundant in tip-growing pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high proportion of the clathrin present is in an assembled state, suggesting a highly dynamic trafficking pathway. Immunofluorescence analysis of pollen tubes revealed that clathrin localises to the plasma membrane at the apex of the pollen tube tip, which is consistent with high levels of clathrin-mediated membrane recycling. The use of these reagents in conjunction with tip-growing pollen tubes has created a unique opportunity to examine the basis for constitutive endocytosis, so that the more complex question of receptor-mediated pathways in plants can also be assessed.